Tuberculosis (TB) has remained a global health problem, despite the availability and widespread use of BCG as a TB vaccine and the existence of effective sterilizing chemotherapy for drug sensitive forms of the disease. In fact, the TB problem has worsened in recent years in many areas of the world. The global HIV epidemic has continued to grow, and it has disproportionately affected resource-limited countries in Africa and Asia. In addition to increased incidence of infection, MDR (Multi Drug Resistant)-TB and XDR (Extensively drug Resistant)-TB have emerged as forms of TB that are significantly more difficult, if not impossible, to treat. Clearly, novel vaccines and novel chemotherapies are urgently needed. We have discovered that a specific Type VII secretion system is responsible for evading killing by innate immunity. An M. smegmatis strain in which these genes have been deleted, designated IKE, has been demonstrated to be safe in at least eight different immunocompromised mouse models. Infection of immunocompetent mice with IKE induces a robust TH1 immune response, which is radically different than that induced by the parental strain, making IKE an attractive vaccine vector. We have shown that the introduction of genes encoding immunodominant TB antigens into IKE creates a strain (IKEPLUS) that can induce bacteriocidal immunity upon challenge with virulent MTB. In this application, we plan to improve IKEPLUS and develop a manufacturing process to make reproducible Good Manufacturing Practices (GMP) batches of a potent IKEPLUS vaccine for human use. We will develop assays to identify biomarkers for the most potent lots. We plan to evaluate the efficacy of this alone and in BCG-primed IKEPLUS-boost regimens to protect against virulent challenges of M. tuberculosis and XDR-M. tuberculosis. PUBLIC HEALTH RELEVANCE: M. tuberculosis strains that are resistant to 2-10 drugs are emerging around the world, causing a worsening of the global TB problem. Novel therapies are urgently needed - particularly, a new and effective vaccine; to this end we have developed a genetically engineered M. smegmatis strain that elicits bacteriocidal immunity against M. tuberculosis. This application will develop the methodologies to optimize this vaccine for use in humans.